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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-March-2010 to 11-March-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: aqueous solution
Details on test material:
- Physical state: Clear yellow liquid
Liquid
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- Expiration date of the lot/batch: 18 December 2010

- Other:
General information: Forms polymeric ions and Al(OH)3 when diluted with water, depending on pH
Density: 1.363 kg/L at 20°C
pH: 1.0 ± 0.5% (20°C)

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 33, 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 33, 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Test compound was soluble in water and water has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9: 60 µg/plate in water for TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 3330 μg/plate and above in the absence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 1000 µg/plate and above and with S9: 3330 µg/plate and above
TA1537: without S9: 1000 µg/plate and above and with S9: 1000 µg/plate and above
TA98: without S9: 1000 µg/plate and above
TA100: without S9: 3330 µg/plate and above and with S9: 5000 µg/plate

Applicant's summary and conclusion

Conclusions:
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the test conditions, the NOTOX substance 202029/A is not mutagenic in the presence and absence of metabolic activation in Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and in Escherichia coli WP2uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP,S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test material at the following concentrations both in the presence and absence of metabolic activation system (rat liver S9-mix) using the plate incorporation method in two independent experiments.

Experiment-1: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in TA 100 and WP2uvrA strains; 33, 100, 333, 1000, 3330 and 5000 µg/plate in TA 1535, TA 1537 and TA 98 strains, without and with 5 % (v/v) S9-mix.

Experiment-2: 33, 100, 333, 1000, 3330 and 5000 µg/plate in all strains, without and with 10 % (v/v) S9-mix.

Vehicle and positive control groups were also included in mutagenicity tests.

The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. In tester strain TA100, toxicity was only observed at dose levels of 3330 and 5000 μg/plate in the absence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Cytotoxicity was observed in all tester strains except in tester strain TA98 in the presence of S9-mix. In the second experiment, cytotoxicity was observed in all tester strains except in the tester strains TA98 in the presence of S9-mix and in WP2uvrA in the absence and presence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation in two independently repeated experiments.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.